Infectious diseases are a major health problem in North America, in part due to the widespread emergence of antibiotic-resistant pathogens. Tuberculosis (TB) is a dramatic example of this recurring threat, with the appearance of highly virulent and multidrug resistant M. tuberculosis(MTB), and the increased prevalence of TB in AIDS patients. The mechanisms of host defense against infection with intracellular parasites such as mycobacteria, and the bacterial strategies underlying long-term survival and replication in host phagocytes remain poorly understood. A better understanding of the host mechanisms of defense against such infections may suggest new strategies for intervention in these diseases. Using a genetic approach to study natural resistance and susceptibility traits in mouse models of human mycobacterial infections, the applicant has identified a new component of the antimicrobial defenses of phagocytes. In the mouse, replication of mycobacteria and other human intracellular pathogens including Salmonella, Leishmania, and Brucella, is controlled by the Bcg locus. The principal investigator cloned Bcg and showed that it encodes a phagocyte specific membrane protein (Nramp1) that shares structural characteristics of known ion transporters and channels. Nramp1 is recruited to the membrane of the phagosome soon after phagocytosis, and the principal investigator has proposed that the substrate of Nramp1 affects intraphagosomal microbial replication. The current proposal has four main goals. The first is to determine if the Nramp1 gene and protein are important for host defense against infection with MTB. For this, the effect of loss of Nramp1 in vivo on replication of MTB in reticuloendothelial organs and on overall mortality, will be evaluated. The second goal is to characterize the effect of Nramp1 association with the mycobacterial phagosome on the biochemical/physiological properties of this organelle, and on the survival of mycobacteria and Salmonella at that site. The third goal is to identify the mechanism of transport and the substrate of Nramp1. The fourth goal is to evaluate the extent of functional homology between the phagocyte-specific Nramp1 protein and the ubiquitously expressed Nramp2 protein. Together, these studies should clarify the mechanism of action of Nramp1 in the antimicrobial defenses of the phagocyte, which may in turn suggest new interventions against TB and other infectious diseases.